The retinotectal system of Xenopus laevis is used as a model system for studying the development of topographically ordered projections in the nervous system. Using in vivo electrophysiological recording and optical imaging techniques, together with molecular manipulations of cellular signaling, we propose to examine the role of cell surface-bound axon guidance molecules ephrin-B1 and EphB2 in regulating the maturation and plasticity of retinotectal synapses after the axons of retinal ganglion cells (RGCs) have made functional synaptic contacts with their targeted tectal cells. The proposed studies are based on our preliminary findings that perturbation of ephrin-B1 reverse signaling in the RGCs affects the maturation of synaptic functions, the dynamics of RGC axon arbors, and the magnitude of activity-induced long-term potentiation (LTP) of retinotectal synapses. In the present project, we propose to further examine the contribution of both ephrin-B1 reverse signaling and EphB2 forward signaling in regulating the maturation and plasticity of retinotectal connections and to determine how this regulation contributes to developmental and visual experience-driven refinement of the receptive field (RF) properties of tectal cells. Selective manipulations of ephrin-B1 and EphB2 signaling will be performed to dissect the modulatory effects of ephrin-B1 vs. EphB2 signaling, by using three different methods, either alone or in combination: (1) acute perfusion of the tectum with the ectodomain fusion protein EphB2-Fc or ephrin-B1-Fc, (2) expression of wild-type or mutated forms of Ephrin-B1 or EphB2 in a few RGCs or tectal cells by electroporation, and (3) global tetracycline-induced transgenic expression of these proteins in either all presynaptic RGCs or all postsynaptic tectal cells. In Aim 1, we will examine the relative contribution of ephrin-B1 reverse signaling and EphB2 forward signaling to the maturation of synaptic functions, identify the pre- and postsynaptic loci of modulation by ephrin-B1/EphB2 signaling, and test the involvement of dynamin-dependent endocytosis and glutamate receptor recruitment in pre- and postsynaptic modulation, respectively. In Aim 2, we will examine the contribution of ephrin-B1/EphB2 signaling to the modulation of LTP and long-term depression (LTD), the postsynaptic mechanisms underlying such modulation, and the role of ephrin-B1/EphB2 signaling in modulating the arbor dynamics of both RGC axons and tectal cell dendrites. In Aim 3, we will examine whether ephrin-B1/EphB2 signaling contributes to the developmental reduction of the RF size, the progressive matching of excitatory and inhibitory RFs, and visual experience- induced direction selectivity in the tectal cell responses to visual stimuli. Together, these in vivo studies offer unique opportunities to address the function of an important set of axon guidance molecules in regulating synapse maturation and plasticity, and to understand the role of trans-synaptic molecular signaling in developmental and experience-driven refinement of neural circuits. PUBLIC HEALTH RELEVANCE: Brain development depends critically on the timely maturation of synaptic connections, a process known to be regulated by both molecular factors coded by the genetic program and neural activities triggered by sensory experience. This project focuses on the function of an important set of molecular factors, ephrin-B1 and EphB2 receptor, in regulating the maturation of neural circuits. Results from the proposed studies will shed light on the mechanisms underlying normal neural circuit development and the potential causes of malformation of neural circuits during development.